Multiparameter immunoprofiling for the diagnosis and differentiation of progressive versus nonprogressive nontuberculous mycobacterial lung disease–A pilot study

Clinical prediction of nontuberculous mycobacteria lung disease (NTM-LD) progression remains challenging. We aimed to evaluate antigen-specific immunoprofiling utilizing flow cytometry (FC) of activation-induced markers (AIM) and IFN-γ enzyme-linked immune absorbent spot assay (ELISpot) accurately identifies patients with NTM-LD, and differentiate those with progressive from nonprogressive NTM-LD. A Prospective, single-center, and laboratory technician-blinded pilot study was conducted to evaluate the FC and ELISpot based immunoprofiling in patients with NTM-LD (n = 18) and controls (n = 22). Among 18 NTM-LD patients, 10 NTM-LD patients were classified into nonprogressive, and 8 as progressive NTM-LD based on clinical and radiological features. Peripheral blood mononuclear cells were collected from patients with NTM-LD and control subjects with negative QuantiFERON results. After stimulation with purified protein derivative (PPD), mycobacteria-specific peptide pools (MTB300, RD1-peptides), and control antigens, we performed IFN-γ ELISpot and FC AIM assays to access their diagnostic accuracies by receiver operating curve (ROC) analysis across study groups. Patients with NTM-LD had significantly higher percentage of CD4+/CD8+ T-cells co-expressing CD25+CD134+ in response to PPD stimulation, differentiating between NTM-LD and controls. Among patients with NTM-LD, there was a significant difference in CD25+CD134+ co-expression in MTB300-stimulated CD8+ T-cells (p <0.05; AUC-ROC = 0.831; Sensitivity = 75% [95% CI: 34.9–96.8]; Specificity = 90% [95% CI: 55.5–99.7]) between progressors and nonprogressors. Significant differences in the ratios of antigen-specific IFN-γ ELISpot responses were also seen for RD1-nil/PPD-nil and RD1-nil/anti-CD3-nil between patients with nonprogressive vs. progressive NTM-LD. Our results suggest that multiparameter immunoprofiling can accurately identify patients with NTM-LD and may identify patients at risk of disease progression. A larger longitudinal study is needed to further evaluate this novel immunoprofiling approach.


Introduction
Nontuberculous mycobacteria (NTM) pathogens are causing more disease burden than tuberculosis (TB) in several regions of the world.The annual incidence and prevalence of NTM disease from 2008 to 2015 increased from 3.13 to 4.73 and 6.78 to 11.70 per 100 000/ year respectively in the United States [1].NTM lung disease (NTM-LD) is the most common presentation accounting for 80-90% of all cases [2].Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MAbsC), are among the most common etiologies of NTM lung disease (NTM-LD) [3][4][5].Many challenges are associated with managing patients with NTM-LD, including suboptimal diagnosis and non-guidelines-based treatments which are often difficult to tolerate.The clinical spectrum of NTM pulmonary infections is also wide, and the relationship between exposure, airway colonization, as well as nonprogressive and progressive infections with clinical and/or radiological disease manifestations, is not well-defined.Symptoms associated with NTM-LD are often non-specific, and the presence of NTM in a single sputum sample does not necessarily equate to active lung disease.Therefore, clinical and radiological findings, as well as microbiological criteria, are critical for the accurate diagnosis of NTM-LD [6][7][8].Moreover, some patients are unable to provide adequate sputum samples, which can also preclude the timely diagnosis of NTM-LD.Importantly, many patients have limited disease severity without clinical and/ or overt radiological signs of progression over time.These patients may not require antimicrobial therapy and are often managed with active airway clearance treatment and close clinical-radiological surveillance [9].When the infection progresses, NTM causes inflammation of the airways and lung tissue which can lead to progressive lung damage and systemic illness.
Currently, there are no diagnostic tests to a priori differentiate progressive from nonprogressive NTM-LD, and the risk factors of disease progression remain unclear.In fact, the clinical assessment of these patients is frequently complicated by the presence of other comorbidities, including pulmonary diseases such as chronic obstructive pulmonary disease (COPD) and bronchiectasis [10].Therefore, the development of non-sputum-based biomarkers and methods that accurately diagnose NTM-LD and reliably identify patients at risk of progressive disease are urgently needed.Such new blood-based NTM diagnostics would facilitate risk stratification and inform the timely individualized management of patients with nonprogressive and progressive forms of NTM-LD [11].
Flow cytometric (FC) detection of activation-induced markers (AIM) in T-cells after ex vivo antigen challenge in peripheral blood mononuclear cells (PBMC) can differentiate the state of infection due to M. tuberculosis [12][13][14][15].We previously reported that combinatory immunoprofiling using FC detection of AIM CD25 + and CD134 + in T-cells following ex vivo antigen challenge in PBMCs can differentiate treated and untreated latent TB infection and might identify patients at risk for future TB reactivation [12].Herein we conducted a pilot study to test whether a similar immunoprofiling approach can accurately identify and differentiate patients with nonprogressive and progressive NTM-LD, which can potentially assist to individualize treatment management for these patients.

Study participants
The study was approved by the Mayo Clinic Institutional Review Board (IRB)(IRB#:09-003253).All study participants signed a written informed consent and were enrolled in Mayo Clinic, Rochester, Minnesota between August 2017 and November 2021.All the methods were carried out in accordance with relevant guidelines and regulations after obtaining approval and recommendations from the Mayo Clinic IRB.This pilot study was part of a prospective, single-center, laboratory technician-blinded larger study to investigate new immune biomarkers in tuberculosis, which includes patients with NTM-LD as control subjects.We recruited NTM-LD patients, including patients with clinical and radiological signs of progressive diseases as well as patients with nonprogressive NTM-LD.We obtained the patient clinical data from their medical records: age, sex, ethnicity, smoking history, usage of immunosuppressive drugs, history of TB and other underlying pulmonary diseases, co-morbidities, radiographical features, BMI, bacterial culture results, pulmonary function and other laboratory test results.We applied the American Thoracic Society and the Infectious Diseases Society of America (ATS/IDSA) guidelines diagnostic criteria for NTM-LD [7].We also included local subjects with negative QuantiFERON In-tube™ (QFT) results and no clinical evidence or radiological signs of NTM-PD as study controls.Nonprogressive NTM-LD was defined as clinical and radiological stability over at least 24 months in patients not treated with antibiotics for NTM-LD.Patients that were determined to have progressive NTM-LD had signs of clinical and/or radiological progression over at least 6 months and/or were judged by their clinician to receive antibiotic treatment for NTM-LD.Subjects under 18 years of age and individuals with latent TB infection (LTBI) were excluded from the study.Other study exclusion criteria included: patients living with HIV, active cancer, autoimmune diseases, or a history of solid organ or hematological transplantation.

PBMC isolation
Peripheral blood from the enrolled study participants was collected in sodium-heparin tubes.PBMCs were isolated using the Ficoll density gradient separation protocol as previously described [12].The viable cells were counted by hemocytometer and cryopreserved in the freezing media (10% DMSO with 90% cosmic calf serum) (Fisher Scientific, Hampton, NH).

PBMCs ex vivo stimulation, immunostaining, and flow cytometry
The cryopreserved PBMCs were thawed and cultured in Rosewell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich, St. Louis, MO).Then the cells were stimulated with costimulatory antibodies (CD28, CD49d -1μg/mL) (BD Bioscience, San Diego, CA) and test peptides/antigens such as ESAT-6/CFP-10 peptides, PPD, MTB300 peptide pool, Candida antigen, and anti-CD3 with specified concentrations as described above.Cells cultured under similar conditions without any stimulation served as a negative (nil) control.The culture plate was incubated for 40 hours at 37˚C with 5% CO 2, and the cells were stained with specific surface markers of anti-human monoclonal antibodies such as CD4 BV650 (BD Bioscience, San Diego, CA), and CD3 APC-AF750, CD8 krome Orange, CD25 APC-AF700, CD134 APC (Beckman Coulter, Brea, CA).Finally, cells were washed and fixed with 0.5% paraformaldehyde and at least 250,000 cells were acquired by BD LSRFortessa (BD Bioscience, San Diego, CA).Files were exported in FCS 3.0 format and the percentage of phenotypic markers was analyzed by Kaluza software (Beckman Coulter, Brea, CA).The background (nil) response was subtracted from the tested antigen stimulations.Considering the cell count, number of tested antigens, reagent cost and that this is a small pilot study, we performed the flow cytometry in a single well manner for each antigenic condition.

Data analysis
Results were compared using the Chi-square test for categorical variables (Fisher exact test for cells with numbers �5).Data are expressed in the tables as numbers and percentages or medians with interquartile ranges (IQR), as appropriate.Non-parametric Kruskal-Wallis ANOVA with Dunn's post-test comparison was used to compare IFN-γ ELISpot, and FC antigen-specific results in controls, nonprogressive and progressive NTM-LD patients.The Mann-Whitney U-test was used to compare the data between the cohorts.Receiver operating characteristic (ROC) analysis was performed to define the diagnostic potential of markers by deriving the ROC area under curve (AUC) sensitivity and specificity at their 95% confidence intervals (CI) and best cut offs to differentiate the study groups.In stimulation experiments, percentage of activated T cells were adjusted by subtracting the unstimulated control value.The statistical difference was considered significant when the P values <0.05.We use Graph-Pad Prism 9.3.1 (GraphPad Software, San Diego, CA) for statistical analysis.

Clinical characteristics of study participants
Ninety-two individuals were screened for study eligibility and 52 were excluded, including 44 subjects with LTBI (Fig 1).We enrolled 22 control subjects and 18 patients of NTM-LD, including 8 patients with progressive disease and 10 patients with nonprogressive NTM-LD that did not require antimicrobial therapy (Tables 1, 2).Controls subjects included 17 women and 5 men with no known history of M. tuberculosis infection and negative QFT results, along with no clinical or radiological signs of active pulmonary disease or NTM-LD.The demographics and baseline characteristics of the study groups were summarized in Table 1.There were significant differences in age between NTM-PD patients and controls, but no significant difference was found in sex, ethnicity, immunosuppression state, or smoking habits.There were no significant differences between nonprogressive and progressive NTM-LD in terms of weight loss, smoking, alcohol habits and co-morbidities.Similarly, we did not find significant differences between the groups in the laboratory assays like CRP, ESR, PFT, sputum and culture tests (Table 2).BMI was significantly lower in the progressive NTM-LD (19.1 kg/m2) compared with nonprogressive NTM-LD (25.5 kg/m2) (Table 2).M. avium was the most common species and nodular bronchiectasis was a frequent radiological feature in both the nonprogressive and progressive cohorts.Among 8 progressive NTM-LD, 6 patients had MAC and 2 had M. abscessus complex isolates.Of the 10 nonprogressive NTM-LD, 7 had MAC and others had either M. abscessus complex, M. chelonae, or M. kansasii species.BACES scores were similar across these two groups but four patients with progressive disease had cavitary lung lesions (Table 2).

IFN-γ ELISpot for differentiating NTM-LD from control subjects
We compared the IFN-γ secreting T cells by ELISpot upon stimulation of PBMC with various antigens to differentiate the NTM infection status.The background (nil) IFN-γ secretion was subtracted from the stimulated PBMCs.The median levels of IFN-γ secreting T cells were significantly higher in NTM-LD patients than that in the control group (Fig 2A -2D).The diagnostic accuracy of ELISpot with these antigens are shown in Table 3 and ROC analysis is depicted in Fig 5A .ELISpot with MTB300 stimulation showed the highest sensitivity of 78.5% (95% CI 49.2-95.3)with 92.3% (95% CI, 63.9-99.8)specificity.ELISpot with the other RD1, PPD, and Candida antigens showed 64.2% (95% CI, 35.1-87.2) sensitivity and 80% (95% CI, 56.3-94.2) specificity.By subtracting the individual RD1 antigen ELISpot results from MTB300 (MTB300-RD1), we found a significantly higher IFN-γ sfu count for NTM-LD patients than controls (Fig 2E).In contrast, none of the ratios of ELISpot results with various antigen conditions of interest including RD1/PPD, MTB300/PPD, MTB300/Candida, PPD/ Candida, showed statistical significance differences between NTM-LD patients and control subjects (Fig 2F -2I).Therefore, these data suggest that MTB300-specific IFN-γ ELISpot has the best diagnostic accuracy in distinguishing NTM-LD from controls compared with other ELISpot methods.

Discussion
The diagnosis of NTM-LD can be delayed or missed as some patients exhibit non-specific symptoms for several months or years before the diagnosis is made, and for some patients, diagnosis comes after the disease has progressed substantially [17,18].Our study findings suggest that a blood-based multiparametric IFN-γ ELISpot and flow cytometric immunoprofiling approach with mycobacterial antigens were not only able to accurately identify patients with NTM-LD, but also able to accurately differentiate patients with progressive from nonprogressive NTM-LD in whom antibiotics were deferred.These study findings are important for treatment management of patients with NTM-LD which remains challenging, often requiring prolonged treatment with three or more antibiotics, and frequently resulting in intolerance to these treatments [7,19].In addition, about 50% of patients with MAC lung disease achieve culture conversion without antibiotics, and others remain clinically and radiologically stable despite the presence of mycobacteria in sputum [20].There is currently no reliable way to a priori differentiate which patients will improve with or without antibiotics.Some of these NTM-LD patients do not produce adequate sputum quality for testing.Therefore, there is an urgent need to develop new non-sputum biomarkers and accurate testing methods not only to improve diagnosis but also to predict disease progression to inform individualized antimicrobial management for the patients who would benefit the most.
Despite recent progress made in developing risk scores to predict mortality associated with NTM-PD, little progress has been made to predict individual disease progression in patients without cavitary and/or severe disease.The BACES score is composed of low BMI (<18.5 kg/ m 2 ), advanced age (�65 years), presence of cavity, elevated ESR, and male sex which can predict mortality in patients with NTM pulmonary disease [16,21].However, most of these were not significantly associated with progressive disease in our study, and may not directly affect the NTM-LD progression.It has been reported that the impact of BMI was inversely related to NTM-PD development [22,23].Consistent with these reports, we also observed that low BMI was significantly associated with progressive NTM-LD (p-0.05).Further, cavitary lesions were significantly associated with progressive NTM-LD (p-0.02) as 4 out of 8 patients showed progressive cavitary lesions that were also associated with low BMI which is consistent with a previous study [23].Sputum culture results and serological markers have limited potential in identifying progressive NTM-LD disease that requires prompt initiation of antibiotics, which is commonly based on poorly defined patient's clinical, radiological and microbiological characteristics.
The current knowledge of the risk of disease progression and predictors remains limited.Recently, a study reported that high serum levels of Krebs von den Lungen-6 (KL-6) was associated with disease progression in MAC lung disease [24].Further, systemic inflammatory markers, history of TB, cough, weight loss, presence of cavity and malnutrition were also reported as predictive for disease deterioration in NTM-LD [21,22,25,26].However, the results are highly heterologous and depend on the study patients' characteristics, the study settings, and lack of controlled clinical trials renders them inconclusive.Further, the number of studies on immune-based biomarkers, particularly antigen-specific T cell assays, for predicting disease progression are sparce [27,28].Here, our pilot study shows that this new strategy using a multiparametric IFN-γ ELISpot and FC immunoprofiling with a panel of mycobacterial antigens has the potential to improve the diagnosis of NTM infections and a priori infer risk of progressive versus nonprogressive NTM-LD, which can potentially assist individualized treatment management.
In contrast with other immunoprofiling studies, we have used both nonspecific mycobacterial antigens of purified protein derivatives (PPD) and more specific TB antigens such as the RD1-derived peptide pool and the MTB300 peptide megapool.Since 80% of proteins with significant homology are shared between PPDs of different species of NTM and Mycobacterium tuberculosis (M.tb), cross-reactive immunoassay responses are not unexpected [29,30].In contrast to crude M. tb lysate, or culture filtrate proteins, MTB300 contains equimolar concentrations of peptides which are efficiently processed and presented by antigen-presenting cells.This MTB300 peptide megapool contains a mixture of 300 M. tb derived T cell epitopes from 90 M. tb proteins that specifically targets a large fraction of M. tb-specific CD4 + and probably CD8 + T cells, which can share epitopes with NTM species [31,32].In our study, the overall response against PPD and MTB300 was higher in patients with progressive versus nonprogressive NTM-LD, possibly representing a higher antigen encounter by CD8 + T-cells in peripheral blood associated with more progressive forms of NTM-LD.
Several previous studies examined the Th1, Th2 and Th17 cellular immune response responses by ELISA and FC in individuals with NTM-LD, although the methodologies employed have varied widely and the findings have been inconsistent [33].Further, a commercially available anti-glycopeptidolipid (GPL)-core immunoglobulin A (IgA) antibody measurement is approved overseas as a diagnostic tool for pulmonary MAC disease [34][35][36].However, little is known about the clinical utility of the test for assessing disease progression in western countries.Interestingly, our multiparametric IFN-γ ELISpot assay with ex vivo stimulations with RD1, PPD, MTB300, and Candida antigens showed no significant difference between control subjects and NTM-LD patient groups and these results align with prior reports [37,38].A recent large retrospective study from China showed that ELISpot was effective in discriminating NTM from pulmonary TB but not between NTM-LD and controls [37].Because of the opposite trends of IFN-γ response with different antigen conditions in NTM-LD and controls, we analyzed the ratio of various combinations of antigen stimulations.Calculation of ratios can eliminate the impact of individual IFN-γ variations on the ELISpot assay, and is also less affected by underlying host immune status [39,40].In our multiparametric IFN-γ ELISpot assay we found that ratios of RD1/PPD, RD1/anti-CD3, and MTB300/ Candida can significantly differentiate nonprogressive and progressive NTM-LD.Therefore, the calculation of antigen ratios seems to be useful to predict disease progression rather than measuring individual antigen specific IFN-γ responses.Overall, our findings suggest that the ratios of immune response to mycobacterial-specific and non-specific antigen stimulations may show other important aspects of host immune response in patients with progressive disease.
In addition, the CD25/CD134 assay has shown greater sensitivity in detecting M. tb-specific immune responses in late-stage HIV compared to QFT-GIT, which suggests that this noncytokine immunoprofiling is relatively less affected by immunosuppression [41,42].It has been demonstrated that the upregulation of CD25/CD134 after RD1 antigen stimulation occurs in active TB patients with or without HIV infection [41].We previously reported that a non-cytokine activation immunoassay strategy was able to differentiate latent TB patients from TB-uninfected controls and potentially infer risk of TB reactivation by predictive modeling [12].Recently, Lindestam Arlehamn CS et al., also reported that MAC-PD patients showed significantly increased MTB300-specific CD134 + PD-L1 + co-expression in T-cells compared to QFT-positive controls [32].In line with these studies, patients with progressive NTM-LD had a higher frequency of CD8 T-cells co-expressing CD25 + CD134 + compared with patients with nonprogressive NTM-LD.Thus, MTB300 specific CD25 + CD134 + expression is an independent factor of disease progression that helps to identify patients requiring treatment initiation.Taken together, these findings suggest that CD25 + CD134 + expression on CD8 + T cells may represent a surrogate peripheral blood marker for unsuccessful control of NTM-LD.
Our study has some limitations.The first being the small number of NTM-LD patients and control subjects, which is explained by the pilot design of this work.Secondly, among 18 NTM-LD patients, most of them had MAC lung disease, and a subgroup analysis of this smaller group of patients did not achieve significance difference between progressive and nonprogressive MAC lung disease.However, two ELISpot ratios (RD1/PPD, RD1/antiCD3) and a FC (CD8 + CD25 + CD134 + [MTB300-nil] T-cells) assay showed an upward trend in progressive MAC lung disease.This subgroup analysis can lead to type II statistical error due to a small sample size.Therefore, our results should be further tested in a larger cohort that includes not only a larger number of patients with MAC lung disease but also other non-MAC NTM species.Third, we were unable to select age-matched controls for each case.The mean age of the patients with NTM-LD was higher than that of the controls, possibly leading to some bias, nevertheless, we did not observe substantial differences in the IFN-γ ELISpot and FC phenotypes with regard to age.Fourth, in the absence of a gold standard test for NTM exposure, we could not completely rule out prior NTM exposures or NTM infection in the control subjects; however, all of them had negative chest X-rays and no clinical symptoms or radiological signs of NTM-LD.Fifth, we examined and compared immunoprofiling data in mostly asymptomatic control individuals but additional testing and validation research work would need to include patients with NTM-LD mimickers such as patients with non-NTM pneumonia and fungal lung infections, as well as patients with non-infectious lung conditions such as pulmonary malignancies, sarcoidosis and other inflammatory lung diseases.Finally, although this study was conducted in a specialized clinic that shares the same referral practice, the physicians' recommendations and patients' preferences may vary, which can influence treatment decisions and timing to initiate antimicrobials for NTM-LD, and thus, influence some of the progressive versus nonprogressive study designations in this retrospective study.

Conclusions
Our pilot study results suggest that PPD-specific CD25 + CD134 + upregulation in T-cells is a potential blood biomarker to accurately diagnose NTM-LD.Further, the percentage of MTB300-specific CD8 + CD25 + CD134 + in CD8 + T-cells and a multiparametric IFN-γ ELISpot assay can also accurately differentiate disease progression in NTM-LD.To our knowledge, this is the first study to use this immunoprofiling approach to differentiate patients with progressive and nonprogressive NTM-LD.Therefore, our findings suggest that our multiparameter diagnostic strategy with FC and IFN-γ ELISpot assays can not only provide a non-sputumbased diagnostic method to accurately identify NTM-LD patients, but also differentiate progressive disease status from nonprogressive NTM-LD.This novel diagnostic strategy could assist with optimal treatment management in NTM-LD.However, prospective and larger studies would be needed to test and validate this novel immunoprofiling approach.

Fig 1 .
Fig 1. Study flowchart of patient inclusion and exclusion criteria.https://doi.org/10.1371/journal.pone.0301659.g001 measured the percentage of CD4 and CD8 T cells expressing co-stimulatory molecules CD25 and CD134 by FC in PBMC after ex vivo antigen challenge with RD1 peptides, PPD and MTB300 peptides in NTM-LD patients and control subjects.The general gating strategy is shown in S1 Fig.The percentages of CD4 + CD25 + CD134 + T cells in a control subject, and nonprogressive and progressive NTM-LD patients are shown in the upper right quadrants in each plot (Fig 3).The percentage of PPD-specific CD25 + CD134 + in CD4 and CD8 T cells was significantly higher in NTM-LD patients compared with control subjects (Fig 4B, 4G

Table 1 . Demographic and clinical characteristics of study participants.
Abbreviations: TB = tuberculosis; NTM = non-tuberculous mycobacteria; IQR = Interquartile range; N/A = not applicable.Values are number (%) or median [IQR].a P <0.0013 for age comparison between groups by Mann-Whitney U test.Otherwise, no statistically significant differences between groups by Fisher's exact test or R x C exact test.

Table 2 . Clinical characteristics of patients with nonprogressive and progressive NTM-LD.
). ROC analysis revealed that PPD-specific CD25 + CD134 + in CD4 T cells had the highest specificity of 91.

Table 2 .
(Continued) [16]eviations: COPD = chronic obstructive pulmonary disease; FEV1 = Forced expiratory volume in the first second, FVC = Forced vital capacity, DLCO = Diffusing capacity for carbon monoxide, TLC = Total lung capacity; IQR = Interquartile range.Values are number (%) or median [IQR].aPvaluesfor comparison between (Fisher's exact test or Mann-Whitney U when appropriate)b BACES score[16]: body mass index, age, cavity, erythrocyte sedimentation rate, and sex c Includes two patients with macrolide-susceptible M. abscessus complex (subsp.massiliense) in the progressive NTM lung disease group, and one patient with cystic fibrosis and macrolide-resistant M. abscessus complex (subsp.abscessus) lung disease without radiological and clinical progression but persistent sputum culture positive results.d Culture conversion included four patients who converted respiratory cultures within 6 months and two patients who converted cultures within 12 months.e Includes two patients with persistent sputum cultures for macrolide-susceptible MAC isolates, one patient with fibrocavitary and bronchiectatic lung disease who further deteriorate and passed away despite intensification of her antibiotic treatment, and another patient with fibronodular and bronchiectatic lung disease who achieved sputum culture conversion after 23 months following treatment intensification.f Antibiotic treatment for NTM at the time of study blood sampling.https://doi.org/10.1371/journal.pone.0301659.t002